X-linked severe combined immunodeficiency (SCID-Xl) is uniformly fatal in the first years of life if left untreated. Hematopoietic stem cell (HSC) gene therapy offers the best therapeutic option for many patients who do not have HLA-matched donors. In SCID-Xl clinical studies gammaretroviral vector proviruses have dysregulated nearby proto-oncogenes including LM02, leading to clonal expansion and in some cases frank leukemia. Thus, safer vector systems that are less likely to transactivate proto-oncogenes are needed for SC1D-X1 gene therapy. FV vectors may be a safer alternative to the gammaretroviral vectors used for SCID- Xl clinical trials. They have a favorable integration profile with respect to integration near proto-oncogenes and a reduced propensity to transactivate nearby genes relative to gammaretroviral and lentiviral vectors. Our long term goal is to develop safer and more effective FV vectors for HSC gene therapy, and to better understand the relative risks of using these vectors. Additionally, we would like to better understand host restriction mechanisms that impact FVs. If host restriction mechanisms can be identified and eliminated from the cells used to produce FV vectors, FV vector titers may be improved. This would reduce the costs of future clinical trials, potentially leading to increased use of FV vectors in the clinic. Our objectives in this application are to establish the relative safety of FV vectors using a novel approach to assess genotoxicity, to develop safer insulated FV vectors, and to improve the efficiency of FV vector production. We will employ an innovative shuttle vector approach that does not rely on PCR-based exponential amplification to better understand potential FV vector genotoxicity. Our proposal is highly integrated with the other program Projects. We will collaborate to test our novel insulated FV SCID-Xl vectors in the mouse (Project 1) and dog (Project 2) SCID-Xl models using this novel shuttle vector approach to generate highly significant pre- clinical data. Our central hypothesis is that FV vector safety and efficiency of production can be improved. The proposed research is significant because it is expected to lead to the use of FV vectors in the clinic for life-threatening diseases including SCID-Xl.

Public Health Relevance

The proposed project is directly related to public health because developing improved methods to assess vector genotoxicity, and developing safer and more effective gene therapy vectors is expected to lead to successful treatment of SCID-Xl and other hematopoietic diseases. The proposed research is thus directly related to the NIH's mission to develop innovative research strategies and apply them to improve human health.